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SpCas9 Plasmids

The CRISPR/Cas system can be implemented in mammalian cells by co-expressing the S. pyogenes Cas9 (SpCas9) nuclease along with the guide RNA. For most applications, we recommend using Cas9 with the single guide RNA (sgRNA), which is derived from a synthetic fusion of the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA; Hsu et al., Nature Biotechnology 2013). We also have an older system using two cassettes to express the CRISPR RNA array and tracrRNA separately (Cong et al., Science 2013). Additionally, we have a plasmid expressing SpCas9 alone without sgRNA. Full references are below.

1. SpCas9 (or SpCas9n, D10A nickase) + single guide RNA: These plasmids contain two expression cassettes, a human codon-optimized SpCas9 or SpCas9n, and the single guide RNA. The vector can be digested using BbsI, and a pair of annealed oligos (design is indicated below) can be cloned scarlessly into the vector before the sgRNA scaffold. The oligos are designed based on the target site sequence (20bp) and need to be followed on the 3' end by a 3bp NGG PAM sequence. We have found that adding an additional guanine nucleotide to the 5’ of 20-bp guide can increase targeting efficiency. SpCas9 and SpCas9n with 2a-Puro and 2a-EGFP are also available.

2. SpCas9 (or SpCas9n, D10A nickase) + CRISPR RNA array + tracrRNA: This plasmid contains three expression cassettes. To target a given site, the plasmid can be digested using BbsI, and a pair of annealed oligos (design is indicated below) can be cloned into the CRISPR array. The oligos are designed based on the target site sequence (30bp) and need to be followed on the 3' end by a 3bp NGG PAM sequence.

Pooled genome-wide human SAM library

SAM can be combined with a human genome-wide library to activate all known coding isoforms form the RefSeq database (23,430 isoforms) for gain-of-function screening. For more information,
please visit the Addgene SAM library page
.

Targeted gene activation using SAM

There are two sets of 3 vectors each available for mammalian endogenous gene activation using SAM:

AAV - In vivo Genome Editing

The Zhang Lab has both single- and dual-vector systems that allow the delivery of Cas9 and sgRNA in vivo via adeno-associated virus (AAV). The single vector system uses a smaller Cas9 from S. aureus (SaCas9) that has been human codon-optimized, and packages both the SaCas9 gene and its single guide RNA (sgRNA) into one plasmid (Ran et al., Nature 2015). The dual vector system uses S. pyogenes Cas9 (SpCas9), using one vector to express SpCas9, and another to express up to three sgRNAs (Swiech et al., Nature Biotechnology 2014). Full references are below.

SaCas9 + single guide RNA: These plasmids contain two expression cassettes, a human codon-optimized SaCas9 driven by either the ubiquitous cytomegalovirus (CMV, PX601 [#61591]) or liver-specific tyroxine binding globulin (TBG, PX602 [#61593]) promoter, and a U6-driven single guide RNA. The vector can be digested using BsaI (This is different from BbsI, which is used in the SpCas9 plasmid series.), and a pair of annealed oligos can be cloned scarlessly into the vector before the sgRNA scaffold. The oligos are designed based on the target site sequence (21-22bp) and need to be followed on the 3' end by NGRRT or NGRRN PAM sequences. We have found that adding an additional guanine nucleotide to the 5’ of 21-bp guide can increase targeting efficiency. These plasmids use the inverted terminal repeats (ITR) from AAV serotype 2.

AAV - Cas9 mouse

CRISPR-Cas9 is a versatile genome editing technology for studying the function of genetic elements. To broadly enable the application of Cas9
in vivo
and
ex vivo
, the Zhang Lab established Cre-dependent and constitutively expressing Cas9 knockin mice (Platt
et al
., Cell 2014). In these mice the CRISPR-Cas9 system can be implemented by delivering Cre and sgRNA to a Cre-dependent mouse or sgRNA to a constitutively Cas9-expressing mouse. Described here are AAV vectors that can be combined with Cas9 in a wide range of applications.
Full references are below.

CRISPR-Cas9 Cre expression vectors for cancer modeling

Using Cas9 mice, Platt
et al
. simultaneously modeled the dynamics of
KRAS
,
p53
and
LKB1
, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector (
AAV-KPL
) in the lung generated loss-of-function mutations in
p53
and
Lkb1
, as well as homology directed repair-mediated
Kras
G12D
mutations, leading to macroscopic tumors of adenocarcinoma pathology. These plasmids as well as a backbone plasmid for cloning new targets are described here.

This plasmid contains two expression cassettes, Renilla luciferase-2A-Cre recombinase and an sgRNA targeted to LacZ, which is not present within the mouse genome. This plasmid is used as a control for AAV-KPL.

This plasmid contains two expression cassettes, Renilla luciferase-2A-Cre recombinase and an sgRNA backbone for cloning new targeted plasmids. The plasmid can be digested using SapI, which will reveal sticky ends to enable the rapid ligation of annealed and phosphorylated oligos designed based on the target site sequence (20bp).

CRISPR-Cas9 Cre expression vectors for genome editing in the brain

Using Cas9 mice Platt et al. demonstrated
in vivo
genome editing in the brain by AAV-mediated expression of an sgRNA targeting the neuronal-specific gene NeuN. As a control they designed an sgRNA targeting LacZ, which is not present in the mouse genome. These plasmids are described here.

This plasmid contains two expression cassettes, Cre recombinase and an sgRNA backbone for cloning new targeted plasmids. The plasmid can be digested using SapI, which will reveal sticky ends to enable the rapid ligation of annealed and phosphorylated oligos designed based on the target site sequence (20bp).

This plasmid enables Cre/loxP recombination and fluorescence assisted sorting of cells and nuclei in addition to sgRNA expression. This plasmid contains two expression cassettes, Cre recombinase-2A-EGFP-KASH and an sgRNA.

This plasmid facilitates Cre/loxP recombination and fluorescence assisted sorting of cells and nuclei in addition to sgRNA expression. This plasmid contains two expression cassettes, Cre recombinase-2A-EGFP-KASH and an sgRNA backbone for cloning new targeted plasmids. The plasmid can be digested using SapI, which will reveal sticky ends to enable the rapid ligation of annealed and phosphorylated oligos designed based on the target site sequence (20bp).